Standard methods for evaluating the target specificity of antimicrobial agents often involve the use of microorganisms with altered expression of selected targets and thus either more resistant or more susceptible to target specific inhibitors. In this study we present an alternative approach that utilizes physiological bypass mutants. The Saccharomyces cerevisiae sphingolipid bypass mutant strain AGD is able to grow without making sphingolipids and importantly, tolerates loss-of-function mutations in the otherwise essential genes for both serine palmitoyltransferase (SPT) and inositol phosphorylceramide (IPC) synthase. We found that strain AGD was > 1000-fold more resistant than the wild-type strain to selective inhibitors of SPT and IPC synthase. In contrast, strain AGD. which due to abnormal composition of the plasma membrane is sensitive to a variety of environmental stresses, was more susceptible than the wild-type to amphotericin 13, voriconazole, and to cycloheximide. We show that in a simple growth assay the AGD strain is an appropriate and useful indicator for inhibitors of IPC synthase, a selective antifungal target. (C) 2003 Elsevier Inc. All rights reserved.